All organisms must protect themselves from the viruses and other molecular invaders that they inevitably encounter in their environments. It was very recently discovered that prokaryotes (archaea and bacteria) have small RNA-based adaptive immune systems, called CRISPR-Cas systems that effectively control invasions of viruses and plasmids. CRISPR-Cas systems function by capturing and incorporating short viral and plasmid sequences within the CRISPR locus of the host genome, processing individual CRISPR (cr)RNAs from the CRISPR locus transcripts, and using the crRNAs to guide effector complexes to destroy the nucleic acids of the invader. CRISPR-Cas based immunity is mediated by numerous and diverse Cas (CRISPR-associated) proteins and a given organism may possess one or more of the nine distinct sets of known CRISPR-Cas immune modules. We currently know very little about how the key steps in the remarkable CRISPR-Cas defense pathway occur for most of these systems. The long-term objective of this proposal is to understand the molecular mechanisms that govern CRISPR-Cas immunity in Pyrococcus furiosus, an established model organism for understanding CRISPR-Cas biology that utilizes three distinct CRISPR-Cas pathways for invader silencing. In this project, we address the molecular basis for invader sequence acquisition and silencing through the following specific aims: Determine the molecular basis for function and the in vivo RNA targeting potential of the Cmr effector complex. Define the organization and function of Cst and Csa crRNPs. Determine how foreign DNA sequences are captured within CRISPR loci. Using a powerful combination of molecular, genetic, and biochemical approaches, we will delineate the mechanism of invader silencing for the effector complexes of each of the three CRISPR-Cas pathways. For each effector complex, we will work to understand the molecular basis of crRNA loading, target nucleic acid interaction, and invader silencing. We will also undertake to gain the first insight into the intriguing process of acquisition of invader sequences by the CRISPR locus in any system by investigating the generation of invader DNA fragments and incorporation into the host genome. The work will define the CRISPR-Cas pathways used by multitudes of prokaryotes to survive viral predation. The knowledge gained in this study will contribute directly to ongoing efforts aimed at exploiting CRISPR-Cas systems to both strengthen domesticated bacteria (used to produce safe food, pharmaceuticals and biofuels) and combat human pathogens and limit the spread of antibiotic resistance. PUBLIC HEALTH RELEVANCE: Researchers recently discovered that bacteria and related organisms rely on a unique immune system to protect themselves from attack by viruses. Our studies will define the molecular mechanisms that underlie this sophisticated and very novel immune system. The discoveries that we make have tremendous potential to be used to protect the bacteria that we depend on to make food products, pharmaceuticals, and biofuels, and to combat the bacteria that cause human disease and the spread of antibiotic resistance.